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Human coronavirus NL63 open reading frame 3 encodes a virion-incorporated N-glycosylated membrane protein.

Müller MA, van der Hoek L, Voss D, Bader O, Lehmann D, Schulz AR, Kallies S, Suliman T, Fielding BC, Drosten C, Niedrig M - Virol. J. (2010)

Bottom Line: Analysis of purified viral particles revealed that ORF 3 protein is incorporated into virions and is therefore an additional structural protein.This study is the first extensive expression analysis of a group 1 hCoV-ORF 3 protein.We give evidence that ORF 3 protein is a structural N-glycosylated and virion-incorporated protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Bonn Medical Centre, Bonn, Germany.

ABSTRACT

Background: Human pathogenic coronavirus NL63 (hCoV-NL63) is a group 1 (alpha) coronavirus commonly associated with respiratory tract infections. In addition to known non-structural and structural proteins all coronaviruses have one or more accessory proteins whose functions are mostly unknown. Our study focuses on hCoV-NL63 open reading frame 3 (ORF 3) which is a highly conserved accessory protein among coronaviruses.

Results: In-silico analysis of the 225 amino acid sequence of hCoV-NL63 ORF 3 predicted a triple membrane-spanning protein. Expression in infected CaCo-2 and LLC-MK2 cells was confirmed by immunofluorescence and Western blot analysis. The protein was detected within the endoplasmatic reticulum/Golgi intermediate compartment (ERGIC) where coronavirus assembly and budding takes place. Subcellular localization studies using recombinant ORF 3 protein transfected in Huh-7 cells revealed occurrence in ERGIC, Golgi- and lysosomal compartments. By fluorescence microscopy of differently tagged envelope (E), membrane (M) and nucleocapsid (N) proteins it was shown that ORF 3 protein colocalizes extensively with E and M within the ERGIC. Using N-terminally FLAG-tagged ORF 3 protein and an antiserum specific to the C-terminus we verified the proposed topology of an extracellular N-terminus and a cytosolic C-terminus. By in-vitro translation analysis and subsequent endoglycosidase H digestion we showed that ORF 3 protein is N-glycosylated at the N-terminus. Analysis of purified viral particles revealed that ORF 3 protein is incorporated into virions and is therefore an additional structural protein.

Conclusions: This study is the first extensive expression analysis of a group 1 hCoV-ORF 3 protein. We give evidence that ORF 3 protein is a structural N-glycosylated and virion-incorporated protein.

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Subcellular localization study of overexpressed hCoV-NL63 ORF 3 protein in Huh-7 cells. Confocal laser scanning microscopy on cells expressing recombinant ORF 3 protein and co-staining with different antibodies for cellular organelles. Left panels: staining with rabbit-anti-ORF 3 serum and anti-rabbit-Cy2 (Dianova). Middle panels from top to bottom: co-staining of cellular organelles with a mouse-anti-ERGIC53 (A), mouse-anti-Golgi 58 K for the Golgi (B), goat-anti-LAMP-1 for trans-Golgi Network (TGN) and Lysosomes (LYS) together with goat (or donkey)-anti-mouse-Cy3 antibodies (C). Right panels show merged pictures where yellow areas represent colocalization. Partial colocalizations can be observed with all organelle markers indicating that the glycoprotein ORF 3 is processed trans-Golgi. Bars indicate 20 μm.
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Figure 3: Subcellular localization study of overexpressed hCoV-NL63 ORF 3 protein in Huh-7 cells. Confocal laser scanning microscopy on cells expressing recombinant ORF 3 protein and co-staining with different antibodies for cellular organelles. Left panels: staining with rabbit-anti-ORF 3 serum and anti-rabbit-Cy2 (Dianova). Middle panels from top to bottom: co-staining of cellular organelles with a mouse-anti-ERGIC53 (A), mouse-anti-Golgi 58 K for the Golgi (B), goat-anti-LAMP-1 for trans-Golgi Network (TGN) and Lysosomes (LYS) together with goat (or donkey)-anti-mouse-Cy3 antibodies (C). Right panels show merged pictures where yellow areas represent colocalization. Partial colocalizations can be observed with all organelle markers indicating that the glycoprotein ORF 3 is processed trans-Golgi. Bars indicate 20 μm.

Mentions: After showing that the ORF 3 protein can be found within the ERGIC compartment in infected cells we were interested in which other cellular compartments an isolated overexpressed ORF 3 protein can be detected. Therefore we transfected Huh-7 cells and stained the ORF 3 protein with the specific antiserum and co-stained different cellular compartments with specific antibodies (mouse-anti-ERGIC53, mouse-anti-Golgi 58 K, goat-anti-LAMP-1 for trans-Golgi/Lysosomes). As shown in Figure 3 the recombinant ORF 3 protein can be detected in all major compartments of the secretory pathway (Figure 3A for ERGIC, 3B for Golgi and 3C for trans-Golgi and lysosomes). These localizations are in concordance with recently published data on the homologous SARS-CoV ORF 3a protein that is responsible for Golgi membrane rearrangement [42].


Human coronavirus NL63 open reading frame 3 encodes a virion-incorporated N-glycosylated membrane protein.

Müller MA, van der Hoek L, Voss D, Bader O, Lehmann D, Schulz AR, Kallies S, Suliman T, Fielding BC, Drosten C, Niedrig M - Virol. J. (2010)

Subcellular localization study of overexpressed hCoV-NL63 ORF 3 protein in Huh-7 cells. Confocal laser scanning microscopy on cells expressing recombinant ORF 3 protein and co-staining with different antibodies for cellular organelles. Left panels: staining with rabbit-anti-ORF 3 serum and anti-rabbit-Cy2 (Dianova). Middle panels from top to bottom: co-staining of cellular organelles with a mouse-anti-ERGIC53 (A), mouse-anti-Golgi 58 K for the Golgi (B), goat-anti-LAMP-1 for trans-Golgi Network (TGN) and Lysosomes (LYS) together with goat (or donkey)-anti-mouse-Cy3 antibodies (C). Right panels show merged pictures where yellow areas represent colocalization. Partial colocalizations can be observed with all organelle markers indicating that the glycoprotein ORF 3 is processed trans-Golgi. Bars indicate 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2819038&req=5

Figure 3: Subcellular localization study of overexpressed hCoV-NL63 ORF 3 protein in Huh-7 cells. Confocal laser scanning microscopy on cells expressing recombinant ORF 3 protein and co-staining with different antibodies for cellular organelles. Left panels: staining with rabbit-anti-ORF 3 serum and anti-rabbit-Cy2 (Dianova). Middle panels from top to bottom: co-staining of cellular organelles with a mouse-anti-ERGIC53 (A), mouse-anti-Golgi 58 K for the Golgi (B), goat-anti-LAMP-1 for trans-Golgi Network (TGN) and Lysosomes (LYS) together with goat (or donkey)-anti-mouse-Cy3 antibodies (C). Right panels show merged pictures where yellow areas represent colocalization. Partial colocalizations can be observed with all organelle markers indicating that the glycoprotein ORF 3 is processed trans-Golgi. Bars indicate 20 μm.
Mentions: After showing that the ORF 3 protein can be found within the ERGIC compartment in infected cells we were interested in which other cellular compartments an isolated overexpressed ORF 3 protein can be detected. Therefore we transfected Huh-7 cells and stained the ORF 3 protein with the specific antiserum and co-stained different cellular compartments with specific antibodies (mouse-anti-ERGIC53, mouse-anti-Golgi 58 K, goat-anti-LAMP-1 for trans-Golgi/Lysosomes). As shown in Figure 3 the recombinant ORF 3 protein can be detected in all major compartments of the secretory pathway (Figure 3A for ERGIC, 3B for Golgi and 3C for trans-Golgi and lysosomes). These localizations are in concordance with recently published data on the homologous SARS-CoV ORF 3a protein that is responsible for Golgi membrane rearrangement [42].

Bottom Line: Analysis of purified viral particles revealed that ORF 3 protein is incorporated into virions and is therefore an additional structural protein.This study is the first extensive expression analysis of a group 1 hCoV-ORF 3 protein.We give evidence that ORF 3 protein is a structural N-glycosylated and virion-incorporated protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Bonn Medical Centre, Bonn, Germany.

ABSTRACT

Background: Human pathogenic coronavirus NL63 (hCoV-NL63) is a group 1 (alpha) coronavirus commonly associated with respiratory tract infections. In addition to known non-structural and structural proteins all coronaviruses have one or more accessory proteins whose functions are mostly unknown. Our study focuses on hCoV-NL63 open reading frame 3 (ORF 3) which is a highly conserved accessory protein among coronaviruses.

Results: In-silico analysis of the 225 amino acid sequence of hCoV-NL63 ORF 3 predicted a triple membrane-spanning protein. Expression in infected CaCo-2 and LLC-MK2 cells was confirmed by immunofluorescence and Western blot analysis. The protein was detected within the endoplasmatic reticulum/Golgi intermediate compartment (ERGIC) where coronavirus assembly and budding takes place. Subcellular localization studies using recombinant ORF 3 protein transfected in Huh-7 cells revealed occurrence in ERGIC, Golgi- and lysosomal compartments. By fluorescence microscopy of differently tagged envelope (E), membrane (M) and nucleocapsid (N) proteins it was shown that ORF 3 protein colocalizes extensively with E and M within the ERGIC. Using N-terminally FLAG-tagged ORF 3 protein and an antiserum specific to the C-terminus we verified the proposed topology of an extracellular N-terminus and a cytosolic C-terminus. By in-vitro translation analysis and subsequent endoglycosidase H digestion we showed that ORF 3 protein is N-glycosylated at the N-terminus. Analysis of purified viral particles revealed that ORF 3 protein is incorporated into virions and is therefore an additional structural protein.

Conclusions: This study is the first extensive expression analysis of a group 1 hCoV-ORF 3 protein. We give evidence that ORF 3 protein is a structural N-glycosylated and virion-incorporated protein.

Show MeSH
Related in: MedlinePlus